KR101785687B1 - Method for detection of target nucleic acid sequence by multiple amplification nested signal amplification - Google Patents

Abstract

The present invention relates to a multiple amplification nested signal amplification (MANSA) method using multiple amplification double signal amplification, and more particularly, to a method for amplifying a target nucleic acid sequence using a dumbbell oligonucleotide, And a method for amplifying and detecting a target nucleic acid sequence by a signal amplification reaction. The present invention is capable of detecting multiple target nucleic acid sequences in a very limited sample without false positives.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting a target nucleic acid sequence by multiple amplification double signal amplification,

The present invention relates to a multiple amplification nested signal amplification (MANSA) method using multiple amplification double signal amplification, and more particularly, to a method for amplifying a target nucleic acid sequence using a dumbbell oligonucleotide, And a method for amplifying and detecting a target nucleic acid sequence by a signal amplification reaction.

Currently, the most common method used by researchers to obtain genetic samples is the polymerase chain reaction method using DNA polymerase. The oligonucleotide used in the polymerase chain reaction is designed to bind to the opposite strand of the template DNA. The above method has an advantage in that only a desired region of a desired gene can be amplified accurately by designing the oligonucleotide capable of binding to a target DNA by arbitrarily regulating the length and base sequence thereof. On the other hand, If the number of target genes to be amplified is large, the same procedure must be repeated.

In order to overcome such disadvantages, methods have been developed in which a polymerase chain reaction is performed by mixing two or more target genes and primers corresponding to respective target genes into one, and in addition, there are many methods for improving annealing specificity of a primer Were developed. For example, one or more of the following can be used: touchdown PCR (Don et al., 1991), hot start PCR (DAquila et al., 1991), Nestit PCR (Mullis and Faloona, 1987) and booster PCR (Ruano et al. have. Yet another approach improves the specificity of PCR using a variety of enhancer compounds, including enhancers that increase the effective binding reaction temperature, DNA binding proteins, commercially available reactants, and the like. However, it is not possible to obtain successful results in all PCR methods and testing these additives under various binding temperature conditions is a time-consuming and time-consuming task. Although the approaches described above contribute somewhat to improving primer annealing specificities, the methods described above are problematic in that problems resulting from primers used in PCR amplification, such as non-specific products and a fundamental solution to high background, It does not. In addition, the number of genes that can be successfully amplified at one time is only 3 to 4, and the disadvantages such as competition and interference effects between the respective genes and amplification of nonspecific products are not improved, so that they are not actually used In fact.

Thus, in order to perform multiple polymerase chain reaction and allele-specific PCR, it is necessary to optimize the conditions of the target gene, which requires a lot of time, effort, and sample consumption. . In order to overcome this problem, a method such as linker PCR or ligation-mediated PCR (Ligation Mediated PCR) (Journal of Clinical Microbiology, 43 (11): 5622-5627, 2005) Were developed. However, in the case of the linker polymerase chain reaction, cross-contamination may occur due to an experimental characteristic in which a part of the product reacted in the first tube is transferred to the second tube, and in the case of the ligation polymerase chain reaction, Complex experimental methods using enzymes have caused difficulties for researchers and only some of them have used these experimental techniques.

Accordingly, it has been required to develop a technology capable of realizing gene amplification inexpensively and simply. In order to meet such a demand, the technique developed is a method in which amplification does not occur until the primer alone is manipulated and the PCR reaction temperature is reached. An example of such a method is the invention disclosed in Korean Patent Registration No. 649165. In this technique, an additional regulator site is inserted in the initial primer. This regulator region is a polydeoxyinosine linker and the inosine constituting the regulator region is a universal nucleotide having a lower Tm value than the general nucleotides G, A, T, and C, Since it is a universal base, the polydeoxyinosine linker forms a "bubble-like structure" at a specific temperature and blocks nonspecific binding of the primer to the target, thereby suppressing nonspecific amplification of the PCR . Although this technique is somewhat less expensive than the conventional techniques described above, the temperature of the binding step in the first cycle (PCR reaction temperature) and the temperature of the binding step in the second cycle There was an inconvenience that it was required to be different. This is to allow priming to participate from the second PCR cycle to the sequence introduced into the primer. Of course, this "application of different temperatures" is not necessarily required, but it may be necessary to apply different temperatures for efficient PCR. Also, in the above-mentioned technique, there is a restriction that a pre-selection avitrilary nucleotide sequence at the 5'-terminal region should be added and this should not be complementary to any position of the target. This results in additional inconvenience and furthermore, it is not possible to be sure of the success of application of the technique when all the gene sequences of the target are unknown. Therefore, it is necessary to develop a new method which is cheaper and easier to realize than the prior art.

Real-time PCR is widely used in that it can measure amplification products in real time and enables more accurate quantitative analysis. The conventional patent documents relating to real-time PCR include U.S. Patent Nos. 5,210,015, 5,538,848 and 6,326,145. The existing real-time PCR method has an advantage of a homogeneous assay method in which amplification and detection are simultaneously performed, but the number of target nucleic acid sequences that can be detected simultaneously is limited due to limitations of fluorescent reporter molecules. Since existing thermocycler capable of detecting real-time target nucleic acid sequences can detect up to 5-plex simultaneously, the number of target nucleic acid sequences that can be detected at the same time is limited, and in order to analyze a large- Time and additional expensive real-time monitoring equipment is required.

The TaqMan probe method (U.S. Patent No. 5,210,015) and the self-fluorescence probe method (U.S. Patent No. 5,723,591), which are typical examples of real-time PCR, have a problem in that a false positive result is caused by non-specific binding of double- , Realistic detection of the 5-plex is difficult to achieve and requires skilled techniques and know-how. Since the conventional real-time PCR method requires simultaneous amplification and detection, high throughput of real-time PCR equipment is limited.

Accordingly, an object of the present invention is to provide a method for amplifying and detecting a target nucleic acid sequence capable of enabling hot-start PCR by suppressing unintended PCR amplification at room temperature.

Another object of the present invention is to provide a method for amplifying and detecting a target nucleic acid sequence capable of suppressing nonspecific amplification in PCR as a result of predominating amplification from a PCR product in comparison with amplification from an initial template upon PCR amplification will be.

It is a further object of the present invention to provide a method and system for detecting multiple target nucleic acid sequences simultaneously with a multiplexing method and a method for detecting multiple target nucleic acid sequences with improved sensitivity using a much smaller amount of probe and sample than conventional real- And a method for detecting the same.

In order to achieve the above object, the present invention provides a method for detecting a target nucleic acid sequence using a dumbbell structure oligonucleotide, comprising the steps of:

(a) amplifying a target nucleic acid sequence by a first amplification reaction using a dumbbell structure oligonucleotide represented by the following general formula:

General formula: 5'-A-B-C-3 '

Wherein A is a 5'-low Tm specific region having a nucleotide sequence that is substantially complementary to a position of the target nucleic acid sequence to be hybridized, the nucleotide sequence comprising a nucleotide sequence substantially complementary to one position of C , B is a cleavage site containing at least three universal bases, and C is a 3'-high Tm specific region having a nucleotide sequence that is substantially complementary to a position of the target nucleic acid sequence to be hybridized, wherein A Wherein the Tm of the A is lower than the Tm of the C and the segment has the lowest Tm among the three segments and the segments A and C comprise a complementary nucleotide sequence Forming a non-base pair structure under conditions that hybridize to the target nucleic acid sequence, Is carried out under conditions that do not cause hybridization by itself, and

(b) amplifying the primary amplification product by a secondary amplification reaction using a primer and a fluorogenic reporter, and detecting a signal from the fluorescence generation reporter, wherein the detection of the signal comprises the step of Indicates presence.

The method of the present invention enables hot-start PCR by suppressing unintended PCR amplification at room temperature, and further amplifies the amplification from the PCR product in comparison with amplification from the initial template upon PCR amplification, Can be suppressed.

In addition, the method of the present invention can rapidly and accurately simultaneously amplify many different genes by a single PCR using a dumbbell structure oligonucleotide as a primer.

Further, the present invention can simultaneously detect a plurality of target nucleic acids in a multiplexing manner, sort and distinguish a plurality of target nucleic acid sequences according to the purpose of analysis, and use much smaller amounts of probes and samples than conventional real-time PCR methods It is possible to detect a plurality of target nucleic acid sequences with improved sensitivity.

According to the method of the present invention, the presence or absence of mutation of various cancer genes or inhibitory genes according to the difference in melting temperature between the amplification double signal amplification and the fusion curve without any additional restriction enzyme treatment or nucleotide sequence analysis can be accurately Can be detected.

Conventional real-time PCR methods require a complex modified primer structure, such as a labeled probe or a hairpin structure, which makes it difficult to design, synthesize or sequence select probes and primers. However, the primers of the present invention are simple and easy to perform real-time PCR since they are used for target amplification as well as signal amplification without additional marker probes or complexly modified primer structures.

Conventional real-time PCR methods are not suitable for multiplex assays because they are difficult to design and optimize primers or probes. On the other hand, the present invention can accurately detect the target nucleic acid sequence according to the difference between the fusion amplitudes of the target amplified double signal amplification and the fusion curve without additional probes or complicatedly modified primer structures in real-time PCR.

Thus, the present method is considered to have improved temporal and cost efficiency in the development of real-time PCR assays.

1 is a schematic diagram showing a process of amplifying and detecting five target nucleic acid sequences using the multiple amplification double signal amplification of the present invention.
2A to 2F show amplification results (amplification curves) of each of the target nucleic acid sequences at various concentrations using the method of the present invention.
FIG. 3 shows the amplification results (amplification curves) obtained by mixing the target nucleic acid sequences at various concentrations and using the method of the present invention.
FIG. 4 shows the result of amplification of various concentrations of target nucleic acid sequences and electrophoresis analysis.
FIG. 5 shows the results of mixing the target nucleic acid sequences at various concentrations, amplifying using the method of the present invention, and performing a fusion curve analysis.
6A to 6F show the results of performing a fusion curve analysis after amplifying each of the target nucleic acid sequences at various concentrations using the method of the present invention.

The present invention enables hot-start PCR by suppressing unintended PCR amplification at room temperature, and also allows amplification from PCR products to be superior to amplification from the initial template upon PCR amplification, resulting in nonspecific amplification in PCR The present invention provides a method for detecting a target nucleic acid sequence.

The inventors of the present invention have made extensive efforts to develop a method for amplifying a large number of genes using only one PCR reaction. As a result, it has been found that when a primer of a gene sequence to be amplified is prepared, When a primer consisting of a dumbbell-structured oligonucleotide having an arbitrary base sequence complementary to a 3 & tilde & 5-bp termini at the terminal and a universal base pair of 3 to 5 bp connecting the two sites is added, It was confirmed that one polymerase chain reaction can rapidly and accurately amplify many different genes simultaneously. The inventors of the present invention have developed a technology for detecting a target nucleic acid sequence through double signal amplification after securing an amplification product by preferentially performing multiplex target amplification using the dumbbell structure oligonucleotide.

Accordingly, the present invention provides a method for detecting a target nucleic acid sequence from a DNA or nucleic acid mixture using multiple amplification nested signal amplification in liquid phase, which will be made clear by the following examples and claims .

In one embodiment of the invention, the present invention provides a method for detecting a target nucleic acid sequence using a dumbbell structure oligonucleotide, comprising the steps of:

(a) amplifying a target nucleic acid sequence by a first amplification reaction using a dumbbell structure oligonucleotide represented by the following general formula:

General formula: 5'-A-B-C-3 '

Wherein A is a 5'-low Tm specific region having a nucleotide sequence that is substantially complementary to a position of the target nucleic acid sequence to be hybridized, the nucleotide sequence comprising a nucleotide sequence substantially complementary to one position of C , B is a cleavage site containing at least three universal bases, and C is a 3'-high Tm specific region having a nucleotide sequence that is substantially complementary to a position of the target nucleic acid sequence to be hybridized, wherein A Wherein the Tm of the A is lower than the Tm of the C and the segment has the lowest Tm among the three segments and the segments A and C comprise a complementary nucleotide sequence Forming a non-base pair structure under conditions that hybridize to the target nucleic acid sequence, Is carried out under conditions that do not cause hybridization by itself, and

(b) amplifying the primary amplification product by a secondary amplification reaction using a primer and a fluorogenic reporter, and detecting a signal from the fluorescence generation reporter, wherein the detection of the signal comprises the step of Indicates presence.

The method of the present invention comprises (a) a first amplification reaction of a target nucleic acid sequence in a biological sample; And (b) a secondary amplification reaction (signal amplification) of the primary amplification product and a detection reaction of the signal.

According to the present invention, only the target nucleic acid sequence is amplified through the first amplification reaction, and the signal is amplified using the amplification product as a template. Thus, the most significant features of the present invention in comparison with the prior art are as follows: By first amplifying only the target nucleic acid sequence and then performing a secondary amplification (simultaneous signal amplification) of the amplification product, by exonuclease activity Signal amplification and real-time monitoring. Based on this, the present invention is named as "Multiple Amplification Nested Signal Amplification" and is abbreviated as "MANSA".

Multiple amplification of the target nucleic acid can be performed according to various primer-associated nucleic acid amplification methods known in the art. Preferably, the amplification of the target nucleic acid is carried out by PCR, and the PCR method is disclosed in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159. The target nucleic acid sequence to be multiplex amplified is DNA or RNA. The nucleic acid molecule may be double-stranded or single-stranded, preferably double-stranded. When the nucleic acid as a starting material is a double-stranded nucleic acid, it is preferable to make the two strands single-stranded or partially single-stranded. Methods for separating the strands include, but are not limited to, heat, alkaline, formamide, urea and glycocalse treatment, enzymatic methods (eg, helicase action) and binding proteins. For example, chain separation can be achieved by heat treatment at a temperature of 80-105 [deg.] C. When mRNA is used as a starting material, a reverse transcription step is required prior to amplification, and in the reverse transcription step, an oligonucleotide dT primer, a random hexamer, or a complementary oligonucleotide of the target nucleic acid sequence hybridized to the poly A tail of the mRNA Is used. The oligonucleotide dT primer consists of dTMPs, and one or more of the dTMPs can be replaced with other dNMPs to the extent that the dT primer can act as a primer. The reverse transcription step can be accomplished by a reverse transcriptase having RNase H activity. When enzymes with RNase H activity are used, careful selection of reaction conditions avoids the individual RNase H cleavage process.

As used herein, the term "oligonucleotide" refers to a linear oligomer of natural or modified monomer or linkages and includes deoxyribonucleotides and ribonucleotides and is capable of specifically hybridizing to a target nucleotide sequence And is naturally present or artificially synthesized. Oligonucleotides are preferably single strands for maximum efficiency in hybridization. Preferably, the oligonucleotide is an oligodeoxyribonucleotide. Oligonucleotides of the present invention can include natural dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), nucleotide analogs or derivatives. Oligonucleotides may also include ribonucleotides. For example, the oligonucleotides of the present invention can be synthesized by the reaction of a backbone modified nucleotide such as a peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoamidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'- 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexy Tolyl DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines wherein the substituents are fluoro, bromo, chloro, iodo-, methyl-, ethyl-, vinyl-, formyl-, 7-deazapurines having C-7 substituents (the substituents being fluoro, bromo, chloro, bromo, chloro, , children Methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, pyridyl-), inosine and diaminopurine.

As used herein, the term "primer " refers to an oligonucleotide in which the synthesis of primer extension products complementary to the nucleic acid chain (template) is induced, i.e. the presence of a polymerizing agent such as a nucleotide and a DNA polymerase, It can act as a starting point for synthesis at suitable temperature and pH conditions. For maximum efficiency of amplification, preferably the primer is single stranded. Preferably, the primer is a deoxyribonucleotide. The primers of the present invention may comprise naturally occurring dNMPs (i.e. dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primers may also include ribonucleotides.

The primer should be long enough to be able to prime the synthesis of the extension product in the presence of the polymerizing agent. The appropriate length of the primer is determined by a number of factors, such as temperature, application, and the source of the primer.

The term "annealing" or "priming" means that the oligodeoxynucleotide or nucleic acid is apposited to the template nucleic acid, which polymerizes the nucleotide to form a complementary nucleic acid molecule to the template nucleic acid or a portion thereof . The term "hybridization" in this specification means to form a double-stranded nucleic acid from a complementary single-stranded nucleic acid. The terms "annealing" and "hybridization" are no different and are used interchangeably herein.

As used herein, the term "probe" is a single-stranded nucleic acid molecule comprising a substantial portion of a target nucleotide sequence.

The dumbbell structure oligonucleotides used in the present invention serve to amplify a target nucleic acid sequence by hybridizing (annealing) to a target nucleic acid sequence and have a structure represented by the following general formula.

General formula: 5'-A-B-C-3 '

Wherein A is a 5'-low Tm specific region having a nucleotide sequence that is substantially complementary to a position of the target nucleic acid sequence to be hybridized, the nucleotide sequence comprising a nucleotide sequence substantially complementary to one position of C , B is a cleavage site containing at least three universal bases, and C is a 3'-high Tm specific region having a nucleotide sequence that is substantially complementary to a position of the target nucleic acid sequence to be hybridized, wherein A Wherein the Tm of the imager A is lower than the Tm of the C, the division site has the lowest Tm among the three sites, and the division site includes the nucleotide sequence complementary to the A and C Forming a non-base pair structure under the condition that the target nucleic acid sequence hybridizes to the target nucleic acid sequence, It is performed under conditions that hybridization solely by the C does not occur.

The dumbbell structure oligonucleotides used in the present invention are divided into a 5'-low Tm specificity portion and a 3'-high Tm specificity portion separated by a segmentation region Lt; RTI ID = 0.0 > type-specific < / RTI > hybridization.

More specifically, when the dumbbell structure oligonucleotide is annealed to the target nucleic acid sequence, the specificity is not determined by the total length of the primer like the conventional primer, but the 5'-low Tm specificity region and the 3'-high Tm Specificity < / RTI > Thus, when a dumbbell structure oligonucleotide is annealed to a target nucleic acid sequence, it is annealed in a different manner than conventional primers, resulting in a significant improvement in annealing specificity.

In the dumbbell structure oligonucleotides used in the present invention, the universal bases located in the above-mentioned segment are deoxyinosine, inosine, 7-diaza-2'-deoxyinosine, 2-aza-2'- '-OMe inosine, 2'-F inosine, and combinations of the above bases, more preferably deoxyinosine, inosine, or 5-nitroindole, and most preferably deoxyinosine. According to a preferred embodiment of the present invention, the segmentation zone comprises a continuous universal base.

In the dumbbell structure oligonucleotides used in the present invention, the length of the 5'-low Tm specificity region is shorter than the 3'-high Tm specificity region. The 5'-low Tm specificity region preferably has a length of 3-15 nucleotides or 3-5 nucleotides. The 3'-high Tm specificity site has 3-30 nucleotides, 5-19 nucleotides, 6-18 nucleotides, or 18-30 nucleotides in length. The segment has a length of 3-5 nucleotides. Preferably, the 5'-low Tm specificity region has a length of 3 to 5 nucleotides, the cleavage site has a length of 3 to 5 nucleotides, and the 3'-high Tm specificity region has a length of 18 to 30 nucleotides.

According to one embodiment of the present invention, the 5'-low Tm specificity region has a Tm of 10-30 ° C. In addition, the 3'-high Tm specificity region has a Tm of 50-65 ° C. The division preferably has a Tm of 3-10 [deg.] C.

According to an embodiment of the present invention, the 3'-high Tm specificity region may have a nucleotide sequence perfectly complementary to the target nucleic acid sequence to be hybridized.

According to one embodiment of the present invention, the annealing of the dumbbell structural oligonucleotide to the target nucleic acid sequence can be carried out at a temperature in the range of 50 ° C to 65 ° C.

According to an embodiment of the present invention, the first amplification of the target nucleic acid sequence can be performed by repeating a process of template-dependent extension reaction including hybridization, extension and denaturation of the dumbbell structure oligonucleotide.

According to one embodiment of the present invention, the amplification reaction can be carried out according to a polymerase chain reaction.

A variety of DNA polymerases can be used in the polymerase chain reaction of the methods of the present invention, including the "Clenow" fragment of E. coli DNA polymerase I, the thermostable DNA polymerase and the bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu) . When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. It is desirable to provide the reaction mixture with a joinder such as Mg2 +, dATP, dCTP, dGTP and dTTP to such an extent that the desired degree of amplification can be achieved.

According to one embodiment of the present invention, the primary amplification of step (a) can be amplified to a point before the severe competition between the amplification products occurs.

According to an embodiment of the present invention, the fluorescence generated reporter used in step (b) may be SYBR Green, SYTO 9, EvaGreen or LCGreen.

According to one embodiment of the present invention, the primer used in step (b) may be a conventional primer (conventional primer) or a different dumbbell structure oligonucleotide. According to one embodiment of the present invention, the primer used in step (b) may be the same dumbbell structure oligonucleotide as the oligonucleotide used in step (a) above.

According to one embodiment of the present invention, the dumbbell structure oligonucleotides used in steps (a) and (b) are identical to each other, and in step (b), primers having high specificity are preferentially oligonucleotide priming oligonucleotide priming may be substantially biased.

According to one embodiment of the present invention, at least one of the oligonucleotides used in steps (a) and (b) may be degraded by the UNG enzyme by including a UTP nucleotide.

According to an embodiment of the present invention, at least one of the oligonucleotides used in the step (a) comprises a UTP nucleotide, and the oligonucleotide is removed by the UNG enzyme after the step (a) So that contamination in the differential amplification can be substantially prevented.

According to an embodiment of the present invention, the step (a) may be carried out in a range of 1 to 25 cycles. According to another embodiment of the present invention, the step (a) may be carried out in a range of 5 to 20 cycles. According to another embodiment of the present invention, the step (a) may be carried out in a range of 10 to 15 cycles. However, the cycle range can be freely modified by those skilled in the art.

According to an embodiment of the present invention, the signal detection in the step (b) may be performed by measuring the fluorescence signal generated from the fluorescent reporter every cycle and plotting the amplification curve on a cycle-by-cycle basis. For example, by applying a predetermined threshold to the amplification curve, it is possible to confirm whether or not the target nucleic acid sequence is present or absent. In addition, the amplification curve can be used to quantitate the target nucleic acid sequence. For example, the amount of the target nucleic acid sequence present in the sample can be confirmed by measuring the Ct (Cycle Threshold) value in the amplification curve. Qualitative and quantitative analysis of such a target nucleic acid sequence is known in the art.

According to an embodiment of the present invention, the signal detection of step (b) may be performed by melt curve analysis. The melting curve analysis is well known in the art. For example, after completing step (b), the melting curve can be plotted by measuring the fluorescence while increasing the temperature at a constant temperature interval. According to one embodiment of the present invention, the melting curve can be generated with a resolution ranging from 0.05 ° C to 0.02 ° C. According to another embodiment of the present invention, the melting curve can be generated with a resolution of less than 0.02 캜.

In the conventional real-time PCR method, amplification and detection must be performed at the same time. For example, since the conventional real-time PCR performs amplification and detection at the same time, the time required for detection takes about 2 hours. However, since the present invention can perform amplification and detection separately, You can get results within 30 minutes by using.

Further, the present invention can reduce the amount of fluorescent double-labeled probe used by using the dumbbell structure oligonucleotide, thereby greatly reducing the analysis cost, reducing the amount of sample to be used and the analysis time, and greatly improving the detection sensitivity .

In addition, since the multi-amplification double signal amplification (MANSA) technique of the present invention can shorten the reaction time and monitor the reaction products in real time, the workload in the laboratory can be reduced and the multiple polymerase chain reaction can simultaneously detect And can be used as a sample to exclude errors that may occur due to false negativity in the case of a minute sample in performing a conventional real-time PCR reaction.

The present invention provides a method for simultaneously detecting various nucleotide variations or SNPs (nucleotide polymorphisms) in a nucleotide sequence. In particular, the present invention provides a method capable of simultaneously detecting mutations at the same base or the same codon in the nucleotide sequence.

Until now, a method capable of simultaneously detecting two or more nucleotide mutations, particularly, two or more mutations in the same base or the same codon, by a simple amplification reaction (for example, PCR) has not been practically developed. The present invention provides a practical method for identifying such simultaneous mutation detection with a simple amplification reaction.

The dumbbell structure oligonucleotide used in the present invention has a 5'-low Tm specificity portion and a 3'-high Tm specificity portion separated by a cleavage site Lt; RTI ID = 0.0 > type-specific < / RTI > hybridization.

The 3'-high Tm specificity region of the dumbbell structure oligonucleotides of the present invention includes corresponding or complementary nucleotides corresponding to nucleotide variations. When the oligonucleotide of the present invention is hybridized with the sense strand of the target nucleotide sequence, the 3'-high Tm specificity region includes a nucleotide complementary to the nucleotide mutation. When the oligonucleotide is hybridized with the antisense strand, the nucleotide corresponding to the nucleotide mutation .

According to a preferred embodiment of the present invention, the nucleotide corresponding to the nucleotide mutation or complementary nucleotide is located at a position separated by 1 to 2 bases from the 3'-terminal or 3'-terminal of the 3'-high Tm specificity region, Preferably, it is located at a site spaced 1-2 bases from the 3'-end of the 3'-high Tm specificity region. Most preferably, a nucleotide corresponding to or complementary to a nucleotide variation is located at the center or around the center of the 3'- and Tm-specific region. For example, when the 3'-high Tm specificity site is separated by 1 to 2 bases from the 3'-terminal, the nucleotide corresponding to the nucleotide mutation or complementary nucleotide is 3'- and the 5'- It is located in a separated place.

In the 3'-high Tm specificity region of the oligonucleotide, the nucleotide corresponding to the nucleotide mutation is located at a position separated by 1 to 2 bases from the 3'-end of the 3'- and Tm-specific region, and the complementary nucleotide is 3 ' - When the 5'-terminal of the high Tm specificity site is also located at a position separated by 1-2 bases, the following advantageous effect is produced.

For example, when a SNP is detected using a common primer, the mutation site is located at the 3'-terminal. In this case, when the base at the 3'-terminal is not annealed to the target sequence (that is, The Tm value is not significantly different. Therefore, even when mismatching occurs, there is a tendency that an amplification reaction occurs due to annealing, resulting in a false positive result. On the other hand, when the mutation site is located at a center, the Tm values are largely different from each other when the mutation site is annealed to the target sequence (that is, when mismatching) Enzymes also catalyze polymerization reactions at mismatched sites, resulting in false positive results.

According to the present invention, the above-described problems of the conventional art can be completely overcome. For example, if a nucleotide corresponding to a nucleotide mutation or a complementary nucleotide is located at the center of the 3'- and Tm-specific region, if mismatching occurs at this position, the 3'- and Tm- , The Tm value of the 3'-high Tm specificity region is greatly reduced as compared with the case where the Tm value of the 3'-high Tm specificity region is matched. In the entire primer structure, mismatching at the 5'- , The thermostable polymerase does not catalyze the polymerization reaction. Thus, when mismatching, false positive results are not generated.

According to another embodiment of the present invention, the present invention provides a method for detecting a target nucleic acid sequence using a primer pair comprising a dumbbell structure oligonucleotide. The method comprises the following steps.

(a) first amplifying a target nucleic acid sequence using a primer pair comprising at least one dumbbell structure oligonucleotide represented by at least one of the following general formulas, wherein said first amplification comprises at least one of primer hybridization, primer extension and denaturation Including two cycles:

General formula: 5'-A-B-C-3 '

Wherein A is a 5'-low Tm specific region having a nucleotide sequence that is substantially complementary to a position of the target nucleic acid sequence to be hybridized, the nucleotide sequence comprising a nucleotide sequence substantially complementary to one position of C , B is a cleavage site containing at least three universal bases, and C is a 3'-high Tm specific region having a nucleotide sequence that is substantially complementary to a position of the target nucleic acid sequence to be hybridized, wherein A Wherein the Tm of the A is lower than the Tm of the C and the segment has the lowest Tm among the three segments and the segments A and C comprise a complementary nucleotide sequence Forming a non-base pair structure under conditions that hybridize to the target nucleic acid sequence, Is carried out under conditions that do not cause hybridization by itself, and

(b) amplifying the primary amplification product by a secondary amplification reaction using a primer pair and a fluorogenic reporter, and detecting a signal from the fluorescence generation reporter, wherein the detection of the signal comprises the step of amplifying the target nucleic acid sequence .

The method of the present invention can be applied to the detection of multiple target nucleic acid sequences.

According to another embodiment of the present invention, the present invention provides a method for detecting a target nucleic acid sequence using two or more primer pairs comprising a dumbbell structure oligonucleotide. The method comprises the following steps.

(a) firstly amplifying two or more target nucleic acid sequences using two or more primer pairs comprising at least one dumbbell structure oligonucleotide represented by at least one of the following general formulas, wherein the first amplification comprises primer hybridization, And at least two cycles of elongation and denaturation:

General formula: 5'-A-B-C-3 '

Wherein A is a 5'-low Tm specific region having a nucleotide sequence that is substantially complementary to a position of the target nucleic acid sequence to be hybridized, the nucleotide sequence comprising a nucleotide sequence substantially complementary to one position of C , B is a cleavage site containing at least three universal bases, and C is a 3'-high Tm specific region having a nucleotide sequence that is substantially complementary to a position of the target nucleic acid sequence to be hybridized, wherein A Wherein the Tm of the A is lower than the Tm of the C and the segment has the lowest Tm among the three segments and the segments A and C comprise a complementary nucleotide sequence Forming a non-base pair structure under conditions that hybridize to the target nucleic acid sequence, Is carried out under conditions that do not cause hybridization by itself, and

(b) amplifying the primary amplification product by a second amplification reaction using two or more primer pairs and a fluorogenic reporter, and detecting a signal from the fluorescence generation reporter, wherein the detection of the signal Indicates the presence of the target nucleic acid sequence.

According to one embodiment of the present invention, the oligonucleotides used in the above steps (a) and (b) include oligonucleotides for amplifying five or more target nucleic acid sequences, for example, five primer pairs (forward and reverse) and / ≪ / RTI > probe.

The two embodiments described above differ only in the number of the oligonucleotide and the target nucleic acid sequence used, and the overall structure thereof is similar to the embodiment described above, so a detailed description is omitted.

The features of the present invention are summarized as follows:

First, unlike the existing real-time PCR method, the present invention is a method of detecting a signal of a target nucleic acid by using an amplification product, and it is possible to effectively detect a target nucleic acid even when using Taq of several tens or hundreds of times less than the conventional method . Therefore, it can provide cheaper products than existing real-time PCR products.

Second, in order to test whether a virus or a pathogen such as a bacterium is infected, a sample according to each test is separately required. That is, a sample for the virus infection test, a sample for the bacterial infection test, and a sample for the fungal infection test are respectively required. In contrast, the present invention simultaneously amplifies a target nucleic acid sequence of a pathogen such as viruses, bacteria and fungi in a single reaction tube, and then uses the amplified product to detect viruses, bacteria, fungi, viruses / bacteria, viruses / Bacteria / fungi, and the like. That is, various tests can be performed using one sample and a small amount of sample.

Third, in the case of conventional real-time PCR, it is dependent on the concentration of the initial target nucleic acid sequence as a method of detecting simultaneously with amplification using a small amount of target nucleic acid sequence. On the other hand, the present invention has a sensitivity higher than that of the conventional method by adding a process of amplifying a signal for a target nucleic acid in a second detection process using a first amplified product. That is, according to the present invention, a sensitivity improving effect similar to nested PCR can be achieved.

Fourthly, in the present invention, a target nucleic acid sequence of a plurality of viruses, bacteria, and molds is simultaneously amplified in one reaction tube, and screening (classification) tests are performed using the amplified products, The amplified product can be used to perform a secondary identification of the pathogen identified as positive for infection (individual discrimination). That is, a screening test to check whether a virus, a bacterium or a fungus is infected is carried out in the first inspection, and if a virus is judged to be positive, it can be easily and quickly identified without any additional amplification reaction. On the other hand, in the conventional method, although screening is performed to obtain virus positive results, amplification of the viral nucleic acid sequence must be repeated. Therefore, it is possible to perform various tests using the amplified product through one amplification process, so that the inspection time and cost necessary for separate amplification are unnecessary.

Fifth, in the present invention, the amplification reaction of the target nucleic acid sequence is performed using general amplification equipment, and the detection process is performed within 30 minutes through real-time detection equipment. Therefore, when amplified products are prepared by using general amplification equipment, samples of 96 samples (if the real-time detection device is 96-well) can be inspected every 30 minutes. On the other hand, the conventional real-time PCR method requires at least 2 hours and 30 minutes for every 96 samples.

The multiple amplification nested signal amplification (MANSA) technique of the present invention having this advantage will be described in detail with reference to examples.

Example 1: Detection of target nucleic acid sequence by the method of the present invention

Genomic DNA of zikavirus was used as a target nucleic acid sequence. Based on the zikavirus genomic DNA, forward and reverse primers having a dumbbell structure were designed.

The primer having the dumbbell structure used in the present invention basically has the structure of the general formula I so as to hybridize with the target nucleic acid sequence with very high specificity. In this primer having the structure of the general formula I, the 5'-low Tm specificity region and the 3'-high Tm specificity region are physically and functionally divided by the cleavage site, and the hybridization specificity of the entire primer structure is 5'- Tm specificity and 3'-high Tm specificity. In addition, the nucleotide corresponding to the nucleotide mutation or hybridized to the mutation is located at the central portion of the 3'- and Tm specific region, so that the difference in Tm value due to mismatch is increased, while DNA synthesis by Taq polymerase during mismatch Respectively. The target nucleic acid sequences and primer sequences used are shown in Table 1 below.

Target nucleic acid sequence 5'-GGGAAGTTCAACGAGCCAAAAAGTCATATATCTGGTCATGATACTGCTGATTGCCCCGGCATACAGCATCAGGTGCATAGGAGTCAGCAATAGGGACTTTGTGGAAGGTATGTCAGGTGGGACTTGGGTTGATGTTGTCTTGGAACATGGAGGTTGTGTTACCGTAATGGCACAGGACAAACCGACTGTCGACATAGAGCTGGTTACAAC-3 ' SEQ ID NO: 1 Forward primer 5'-GCAATIII GGTCATGATACTGCTGATTGC-3 ' SEQ ID NO: 2 Reverse primer 5'-GCAATIII CCACAAAGTCCCTATTGC-3 ' SEQ ID NO: 3

(Final concentrations of 1 ng, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, 100 gg and 10 gag) were mixed with 5 pmoles of primers and 20 μl final volume containing 10 μl of Type IT HRM master mix in a thermocycler , PerKinelmer). The reaction mixture was denatured at 95 ° C for 10 minutes, and the reaction was repeated 15 times at 95 ° C for 30 seconds, 65 ° C for 60 seconds, and 72 ° C for 60 seconds, followed by final reaction at 72 ° C for 5 minutes.

The tube containing the reaction mixture was then placed in a real-time thermocycler (Rotogene Q, Qiagen); The reaction mixture was denatured at 95 ° C. for 10 minutes, and the procedure was repeated 30 times at 95 ° C. for 10 seconds, 65 ° C. for 15 seconds, and 72 ° C. for 25 seconds to detect an increase in final fluorescence signal. And a signal generated in each cycle by a predetermined time interval was detected.

The results are shown in Fig.

As shown in Fig. 2, no fluorescence signal amplification of the target nucleic acid sequence was observed in a trace amount template. These results indicate that signal amplification does not occur by hybridization of the target template with the primer of the present invention or by cleavage of the single stranded primer of the present invention. On the other hand, fluorescence signals of target nucleic acid sequences of up to 1 fg were confirmed by the concentration of the target nucleic acid sequence. Thus, without amplification of the target nucleic acid sequence, repetition of denaturation, hybridization, cleavage and extension using the primers of the present invention is sufficient to generate the target fluorescent signal, thereby enabling detection of the target nucleic acid sequence by real-time signal amplification do.

Example 2: PCR sensitivity of the method of the present invention

Real-time PCR sensitivity using the primers of the present invention was confirmed by detecting the target nucleic acid sequence of the Zicca virus gene. As shown in FIG. 3, when real-time PCR was performed using Zikavirus genomic DNA diluted serially starting from 1 ng, the target nucleic acid sequence was detected even when diluted to 10 fg.

The results of analysis of the amplified products by electrophoresis analysis are shown in FIG. As shown in FIG. 4, it was confirmed that an amplification product was generated in an amount proportional to the concentration of the initial target nucleic acid sequence.

Example  3: Primer  Analysis of melting temperature curve

The present inventors further confirmed whether the primers of the present invention can generate a sufficient signal to detect a target nucleic acid sequence when repeating denaturation, hybridization, cleavage and extension under real-time PCR reaction conditions by DNA concentration.

To test this evaluation, the same template and primer used in Example 1 were used for real time target signal amplification. Real-time target signal amplification was performed with 20 μl final volume containing 5 pmoles of primers and 10 μl of Type IT HRM master mix at various concentrations of DNA (final concentrations 1 ng, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, ; The tube containing the reaction mixture was placed in a real-time thermocycler (Rotogene Q, Qiagen); Real-time PCR was performed using 1 μl of each of the obtained PCR products as a sample using a Type-it HRM PCR kit (Qiagen Inc., Germantown, MD, USA). About 10ul of 2X Type-it HRM PCR Master Mix, 0.1ul of APC primer mixture, and 9ul of distilled water were added and real-time PCR was performed. The Type-it HRM PCR mastermix contains EvaGreen as a fluorescent dye.

Real-time polymerase chain reaction was performed at 95 ° C for 10 sec, 65 ° C for 15 sec, and 72 ° C for 20 sec using a Rotor-gene Q (Qiagen Inc., Germantown, MD, USA) . After the final polymerase chain reaction, fluorescence intensity of the amplified product was observed by measuring the fluorescence wavelength at 510 nm while increasing the temperature from about 77 to 89 degrees at a rate of about 0.2 degree per second.

As shown in Fig. 5, the fluorescence signal of the target nucleic acid sequence was not amplified in a trace amount of the template, and the melting temperature curve was not observed. These results indicate that signal amplification does not occur by hybridization of the target template with the primer of the present invention or by cleavage of the single stranded primer of the present invention. On the other hand, the fluorescence signal of the target nucleic acid sequence up to 1 fg was confirmed by the template concentration, and the melting temperature curve was confirmed based on the fluorescence signal. Also, as shown in Fig. 6, the melting temperature curve analysis by the template concentration was possible in the target nucleic acid sequence up to 1 fg.

 While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> DIOGENE INC. <120> Method for detection of target nucleic acid sequence by multiple          진화 <130> P16-1525 <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 210 <212> DNA <213> Artificial Sequence <220> <223> genomic DNA of zika virus <400> 1 gggaagttca acgagccaaa aagtcatata tctggtcatg atactgctga ttgccccggc 60 atacagatc aggtgcatag gagtcagcaa tagggacttt gtggaaggta tgtcaggtgg 120 gacttgggtt gatgttgtct tggaacatgg aggttgtgtt accgtaatgg cacaggacaa 180 accgactgtc gacatagagc tggttacaac 210 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <220> <221> misc_feature <222> (6) <223> n is deoxyinosine <400> 2 gcaatnnngg tcatgatact gctgattgc 29 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <220> <221> misc_feature <222> (6) <223> n is deoxyinosine <400> 3 gcaatnnncc acaaagtccc tattgc 26

Claims (32)

A method for detecting a target nucleic acid sequence using a dumbbell structure oligonucleotide, comprising the steps of:
(a) annealing at a temperature in the range of 50 ° C to 65 ° C using a dumbbell-structured oligonucleotide represented by the following formula and subjecting the target nucleic acid sequence to hybridization, extension and denaturation using the dumbbell-structure oligonucleotide Amplifying to a point prior to the occurrence of severe competition between amplification products in the range of 5 to 20 cycles by the first amplification reaction of the polymerase chain reaction repeating the process of the template-dependent extension reaction,
General formula: 5'-ABC-3 '
Wherein A is a 5'-low Tm specific region having a nucleotide sequence that is substantially complementary to a position of the target nucleic acid sequence to be hybridized, the nucleotide sequence comprising a nucleotide sequence substantially complementary to one position of C , B is a cleavage site containing a consecutive sequence of nucleotides containing at least three deoxyinosine as a universal base, and C is a nucleotide sequence having a nucleotide sequence substantially complementary to a position of the target nucleic acid sequence to be hybridized A 3'-high Tm specificity region comprising a nucleotide sequence substantially complementary to one position of said A, said Tm of said A being 10 ° C to 30 ° C lower than the Tm of said C of 50 ° C to 65 ° C, The division site has the lowest Tm of 3 占 폚 -10 占 폚 among the three sites, and the division sites are A and C Wherein the length of the 5'-low Tm specificity region is shorter than the length of the 3'-high Tm specificity region, and the 5'-low Tm specificity region Wherein the 3'-high Tm specificity region has a length of 3 to 5 nucleotides, the division region has 3 to 5 nucleotide lengths and the 3'-high Tm specificity region has 18 to 30 nucleotide lengths, Characterized in that the hybridization is carried out under conditions that hybridization by C alone does not occur, and
(b) contacting the primary amplification product with a primer comprising the same dumbbell structural oligonucleotide as the oligonucleotide used in step (a) above and a fluorogenic reporter comprising SYBR Green, SYTO 9, EvaGreen or LCGreen, Amplifying the amplified product by a secondary amplification reaction and detecting a signal from the fluorescence generation reporter by melt curve analysis generated at a resolution in the range of 0.05 ° C to 0.02 ° C, Indicates the presence of the target nucleic acid sequence.
A method for detecting a target nucleic acid sequence using a dumbbell structure oligonucleotide, comprising the steps of:
(a) first amplifying a target nucleic acid sequence using a primer pair comprising at least one dumbbell structure oligonucleotide represented by at least one of the following general formulas, wherein the first amplification comprises hybridization with the dumbbell structure oligonucleotide, extension And amplification to a point prior to the occurrence of severe competition between the amplification products in the range of 5 to 20 cycles by the first amplification reaction of the polymerase chain reaction repeating the process of the template-dependent extension reaction including the denaturation step:
General formula: 5'-ABC-3 '
Wherein A is a 5'-low Tm specific region having a nucleotide sequence that is substantially complementary to a position of the target nucleic acid sequence to be hybridized, the nucleotide sequence comprising a nucleotide sequence substantially complementary to one position of C , B is a cleavage site containing a consecutive sequence of nucleotides containing at least three deoxyinosine as a universal base, and C is a nucleotide sequence having a nucleotide sequence substantially complementary to a position of the target nucleic acid sequence to be hybridized A 3'-high Tm specificity region comprising a nucleotide sequence substantially complementary to one position of said A, said Tm of said A being 10 ° C to 30 ° C lower than the Tm of said C of 50 ° C to 65 ° C, The division site has the lowest Tm of 3 占 폚 -10 占 폚 among the three sites, and the division sites are A and C Wherein the length of the 5'-low Tm specificity region is shorter than the length of the 3'-high Tm specificity region, and the 5'-low Tm specificity region Wherein the 3'-high Tm specificity region has a length of 3 to 5 nucleotides, the division region has 3 to 5 nucleotide lengths and the 3'-high Tm specificity region has 18 to 30 nucleotide lengths, Characterized in that the hybridization is carried out under conditions that hybridization by C alone does not occur, and
(b) contacting said primary amplification product with a primer pair comprising the same dumbbell structural oligonucleotide as the oligonucleotide used in step (a) above and a fluorogenic reporter comprising SYBR Green, SYTO 9, EvaGreen or LCGreen ) Amplified by a second amplification reaction and detecting a signal from the fluorescence generating reporter by melt curve analysis generated at a resolution in the range of 0.05 to 0.02 ° C, The detection indicates the presence of the target nucleic acid sequence.
A method for detecting two or more target nucleic acid sequences using dumbbell structure oligonucleotides, comprising the steps of:
(a) first amplifying two or more target nucleic acid sequences using two or more primer pairs comprising a dumbbell structure oligonucleotide represented by at least one of the following general formulas, Amplification to the point in time before significant competition between amplification products occurs in the range of 5 to 20 cycles by the first amplification reaction of the polymerase chain reaction which repeats the process of the template-dependent extension reaction including nucleotide hybridization, extension and denaturation process :
General formula: 5'-ABC-3 '
Wherein A is a 5'-low Tm specific region having a nucleotide sequence that is substantially complementary to a position of the target nucleic acid sequence to be hybridized, the nucleotide sequence comprising a nucleotide sequence substantially complementary to one position of C , B is a cleavage site containing a consecutive sequence of nucleotides containing at least three deoxyinosine as a universal base, and C is a nucleotide sequence having a nucleotide sequence substantially complementary to a position of the target nucleic acid sequence to be hybridized A 3'-high Tm specificity region comprising a nucleotide sequence substantially complementary to one position of said A, said Tm of said A being 10 ° C to 30 ° C lower than the Tm of said C of 50 ° C to 65 ° C, The division site has the lowest Tm of 3 占 폚 -10 占 폚 among the three sites, and the division sites are A and C Wherein the length of the 5'-low Tm specificity region is shorter than the length of the 3'-high Tm specificity region, and the 5'-low Tm specificity region Wherein the 3'-high Tm specificity region has a length of 3 to 5 nucleotides, the division region has 3 to 5 nucleotide lengths and the 3'-high Tm specificity region has 18 to 30 nucleotide lengths, Characterized in that the hybridization is carried out under conditions that hybridization by C alone does not occur, and
(b) contacting the primary amplification product with at least two primer pairs comprising the same dumbbell structural oligonucleotide as the oligonucleotide used in step (a) and a fluorescence generating reporter comprising SYBR Green, SYTO 9, EvaGreen or LCGreen amplifying by a second amplification reaction using a fluorogenic reporter and detecting a signal from the fluorescence generating reporter by melt curve analysis generated at a resolution in the range of 0.05 ° C to 0.02 ° C, The detection of the signal indicates the presence of the target nucleic acid sequence.
delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete 4. The method according to any one of claims 1 to 3, wherein step (a) is carried out in a range of 10 to 15 cycles.
4. The method of claim 3, wherein the oligonucleotides used in steps (a) and (b) are oligonucleotides for amplifying five or more target nucleic acid sequences.
delete delete 4. The method according to any one of claims 1 to 3, wherein the melting curve is generated at a resolution of less than 0.02 占 폚.

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